Hypothermic storage of isolated human hepatocytes: A comparison between University of Wisconsin solution and a hypothermosol platform
ABSTRACT Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4 degrees C for 24-72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5'-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.
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- "The EC- and UW-solutions could, for example, preserve human hepatocytes under hypothermic conditions (e.g. +4°C) for 24 to 72 h , . Here we examined whether the performance of a cell-preservation solution is improved by addition of fish antifreeze protein (AFP), for which a general cell-membrane protection ability has been recognized in the last two decades . "
ABSTRACT: It is sometimes desirable to preserve mammalian cells by hypothermia rather than freezing during short term transplantation. Here we found an ability of hypothermic (+4°C) preservation of fish antifreeze protein (AFP) against rat insulinoma cells denoted as RIN-5F. The preservation ability was compared between type I-III AFPs and antifreeze glycoprotein (AFGP), which could be recently mass-prepared by a developed technique utilizing the muscle homogenates, but not the blood serum, of cold-adapted fishes. For AFGP, whose molecular weight is distributed in the range from 2.6 to 34 kDa, only the proteins less than 10 kDa were examined. The viability rate was evaluated by counting of the preserved RIN-5F cells unstained with trypan blue. Significantly, either AFPI or AFPIII dissolved into Euro-Collins (EC) solution at a concentration of 10 mg/ml could preserve approximately 60% of the cells for 5 days at +4°C. The 5-day preserved RIN-5F cells retained the ability to secrete insulin. Only 2% of the cells were, however, preserved for 5 days without AFP. Confocal photomicroscopy experiments further showed the significant binding ability of AFP to the cell surface. These results suggest that fish AFP enables 5-day quality storage of the insulinoma cells collected from a donor without freezing.PLoS ONE 09/2013; 8(9):e73643. DOI:10.1371/journal.pone.0073643 · 3.23 Impact Factor
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- "Trolox is also known to have a significant iron-chelating capacity ; iron chelators have been found to be one of the key elements to enable high attachment efficiency of preserved cells after long term hypothermic storage of rat hepatocytes [27,28]. The importance of Trolox for cold storage of human hepatocytes beyond 48 hours was shown by Ostrowska and co-workers  where they compared HTS-FRS against UW and HTS-Base (without Trolox) and found that HTS-FRS was the only solution to prevent damages beyond 48 hours. While ROS generation during short static cold storage at +4oC has been shown to be low  and the lipid peroxidation to be slow , similar data does not exist for long storage during cold storage or supercooling. "
ABSTRACT: Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.PLoS ONE 07/2013; 8(7):e69334. DOI:10.1371/journal.pone.0069334 · 3.23 Impact Factor
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ABSTRACT: The donor organ shortage is the largest problem in transplantation today and is one where organ preservation technology has an important role to play. Static storage of solid organs, especially of the kidney, continues to be the most common method employed for storage and transport of organs from deceased donors. However, the increase in organs obtained from expanded criteria donors and donors with cardiac death provide new challenges in crafting effective preservation methods for the future. This article reviews the current status of static hypothermic storage methods and discusses potential avenues for future exploitation of this technology as the available organ pool is expanded into the more marginal donor categories.Cryobiology 07/2010; 60(3-60). DOI:10.1016/j.cryobiol.2009.06.004 · 1.59 Impact Factor